Cellulose binding fusion proteins for immobilization and purification of polypeptides

ABSTRACT

A fusion protein is prepared containing a polypeptide such as an enzyme and an amino acid sequence having a substrate binding region of a polysaccharidase such as cellulase that has essentially no polysaccharidase activity. By contacting the fusion protein with an affinity matrix containing a substrate such as cellulose for the cellulase substrate binding region, the substrate binding region binds to the affinity matrix to immobilize the polypeptide. The polypeptide can be purified by separating the fusion protein or polypeptide from the affinity matrix.

INTRODUCTION

1. Technical Field

This invention relates to novel polypeptide compositions, includingchimeric polypeptides capable of binding to a polysaccharide matrix, andmethods for their preparation using recombinant DNA techniques.

2. Background

Production of foreign proteins by expression in microbial systems maybecome a significant source of high value, medically important proteins.Purification and recovery of recombinant proteins are majorconsiderations in the design of a fermentation process. Whiletraditional means of protein purification can be used to isolate aproduct, improved means include the use of fusion proteins. Fusionproteins can be purified by affinity chromatography, the desiredcomponent of the fusion protein being purified by virtue of its covalentattachment to a polypeptide which binds to an affinity matrix. As anexample, fusion proteins comprising a polypeptide of interest fused toβ-galactosidase be purified using aρ-amino-phenyl-β-D-thiogalactoside-Sepharose column. Such a method hasbeen used for purification of immunogenic polypeptides such as viralantigens. Staphylococcal protein A can also be used for affinitypurification of fusion proteins by virtue of its specific binding to theFc portion of immunoglobulins.

In addition to purification, recovery of the original components fromthe fusion is often desirable. Both chemical and biological methods havebeen devised to cleave fusion proteins into their component polypeptidesor segments. Introduction of acid-labile aspartyl-proline linkagesbetween the two segments of a fusion protein facilitates theirseparation at low pH. The major requirement of this system is that thedesired segment of interest is not acid-labile. Fusion proteinscomprising hormones such as insulin and somatostatin have been cleavedwith cyanogen bromide, which is specific for the carboxyl side ofmethionine residues to release the desired hormone. This method is notsuitable when the desired protein contains methionine residues.

Cleavage of fusion proteins by site-specific proteolysis has also beeninvestigated. Fusion proteins into which a chicken pro α-2 collagenlinker was inserted could be specifically degraded by purified microbialcollagenase to release the components of the fusion protein Othermethods for purification and recovery of a desired recombinant proteininclude construction of a poly-arginine tail at the carboxyterminus ofthe protein. The arginine residues increase the overall basicity of theprotein which facilitates purification of the desired protein by ionexchange chromatography. Subsequent removal of the poly-arginine tail bycarboxypeptidase B regenerates the desired protein and allowspurification from basic contaminants due to the reduction in pI of thedesired protein.

It is of interest to develop a rapid and inexpensive method forpurification or immobilization of a desired protein. Carbohydratepolymers such as cellulose are plentiful and inexpensive. Furthermore, avariety of enzymes bind specifically to carbohydrate polymers. It wouldtherefore be of interest to prepare fusion proteins comprising at leastthe carbohydrate polymer-binding portion of such an enzyme as a meansfor immobilizing and/or purifying the fusion protein.

Relevant Literature

The affinity of cellulases for cellulose have been used for theirpurification (Boyer et al., Biotechnol. Bioeng. (1987) 29:176-179;Halliwell et al., Biochem. J. (1978) 169:713-735; Mart'yanov et al.,Biokhimiya (1984) 19:405-104; Nummi et al., Anal. Biochem. (1981)116:137-141; van Tilbeurgh et al., FEBS Letters (1986) 204:223-227).Several cellulase genes from Cellulomonas fimi have been cloned intoEscherichia coli (Whittle et al., Gene (1982) 17:139-145; Gilkes et al.,J. Gen. Microbiol. (1984) 130:1377-1384). Binding to Avicel(microcrystalline cellulose) has been used for purification of bothnative (Gilkes et al., J. Biol. Chem. (1984) 259:10455-10459) andrecombinant enzymes (Owolabi et al., Appl. Environ. Microbiol. (1988)54: 518-523). A bifunctional hybrid prtein which binds maltose has beendescribed. Bedouelle et al., Eur. J. Biochem. (1988) 171:541-549.

Two of the C. fimi cellulases, an exoglucanases (Cex) and anendoglucanase (CenA), have been characterized and their genes, cex andcenA, have been sequenced (Wong et al., Gene (1986) 44:315-324; O'Neillet al., Gene (1986) 44:325-330). Predicted amino acid sequences showevidence of domain structure for these enzymes (Warren et al., PROTEINS:Structure, Function, and Genetics (1986) 1:335-341). Domain structureshave also been observed in other cellulases (Teeri et al., Publications(1987) 38: Technical Research Centre of Finland; Teeri et al., Gene(1987) 51:43-52) and separation of domains by proteolytic cleavage hasgiven some insight into domain function (Langsford et al., FEBS Letters(1987) 225: 163-167; Tomme et al., Eur. J. Biochem. (1988) 170:575-581;van Tilbeurgh et al., FEBS Letters (1986) 204:223-227). A serineprotease found in C. fimi culture supernatants (Langsford et al., J.Gen. Microbiol. (1984) 130:1367-1376) has been shown to cleavesubstrate-bound recombinant CenA and Cex, releasing catalytically-activefragments with greatly reduced affinity for cellulose (Langsford et al.,FEBS Letters (1987) 225:163-167). The remaining fragments correspond tothe irregular regions of low charge density in both enzymes and arebelieved to constitute the cellulose-binding domains of the enzymes.

SUMMARY OF THE INVENTION

Methods and compositions are provided for preparing a fusion proteincapable of binding to a polysaccharide matrix. The fusion proteincomprises at least the substrate binding region of a polysaccharidase.The fusion protein is prepared by transforming into a host cell a DNAconstruct comprising a fragment of DNA encoding at least the substratebinding region of a polysaccharidase gene ligated to a gene encoding apolypeptide of interest and growing the host cell to express the fusedgene. The resulting fusion protein readily binds to a solid supportcomprising a substrate for the polysaccharidase. The composition can beused to prepare a polysaccharide matrix comprising any of a variety ofpolypeptides of interest or in a method for purifying either the fusionprotein or the polypeptide of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows construction of Cex-expressing plasmids pEC-1.1, andpUC12-1.1cex. The functional orientations of the gene coding forβ-lactamase (Ap^(r)), Cex (a cross hatch square) and the promoters forlac are indicated by arrows. Restriction sites: B=BamHI; E=EcoRI;H3=HindIII; S=SalI.

FIG. 2 shows obtaining pUC12-1.1(PTIS).

FIG. 3 shows construction of pUCEC.2.

FIGS. 4A and 4B show plasmid construction for obtaining pUCEC2.

FIG. 5-1 and 5-2 show construction of pE01. Sp=SphI; Ss=hybrid SmaI;Sa=SalI; PS=PstI; ABG=β-galactosidase gene Cex exogluconase gene;SBD=substrate binding domain; PT=proline-threonine box hatched boxmultiple cloning site.

FIG. 6 is a schematic diagram of linearized pUC12-1.1cex (PTIS) showingrelevant restriction sites.

FIG. 7 is a schematic diagram of a reusable fermentor-immobilizationcolumn set up for the hydrolysis of cellulosic materials to glucose.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Novel compositions comprising fusion proteins in which at least thesubstrate binding portion of a cellulase is fused to a protein ofinterest, as well as methods for their preparation, are provided. Thecompositions may be prepared by transforming into a host cell a CNAconstruct comprising at least a fragment of DNA encoding the substratebinding region of a polysaccharidase gene ligated to a DNA sequenceencoding the peptide of interest and growing the host cell to expressthe fused gene. The host cell may be either a eukaryotic or aprokaryotic cell. The fusion proteins provide for a wide variety ofapplications including purification of the protein of interest,immobilization of the protein of interest, and preparation of solidphase diagnostics, as well as any other applications where a means ofbinding a compound of interest to a polysaccharide matrix is desired.

Novel polypeptide compositions will for the most part have the followingformula:

    SBR--MR--X

wherein:

SBR can be either the N-terminal or the C-terminal region of the subjectpolypeptide and is characterized as having from 108 to 134 amino acidswhich correspond to a consecutive sequence of amino acids from at leastthe substrate binding region of a polysaccharidase;

MR is the middle region, and may be a bond; short linking group of from2 to 30 carbon atoms, or have from about 2 to about 20 amino acids. Theregion may include an amino acid sequence providing for specificcleavage of the fusion protein, usually a sequence corresponding to thatrecognized by a proteolytic enzyme of high specificities such as an IgA₁protease; and

X can be either the N-terminal or the C-terminal region and may be anypeptide of interest. It is characterized as having up to the entiresequence of a polypeptide of interest, or a fragment thereof, and may bean enzyme, a hormone, an immunoglobulin, a dye, etc.

Preparation of Fusion Proteins

The techniques used in isolating a cellulase gene are known in the art,including synthesis, isolation from genomic DNA, preparation from cDNA,or combinations thereof. The various techniques for manipulation of thegenes are well known, and include restriction, digestion, resection,ligation, in vitro mutagenesis, primer repair, employing linkers andadapters, and the like (see Maniatis et al., Molecular Cloning, ColdSpring Harbor Laboratory, Cold Spring Harbor, New York, 1982).

Generally, the method comprises preparing a genomic library from anorganism expressing a cellulase with the desired characteristics.Examples of such cellulases are those obtainable from strains belongingto the species of Cellulomonas fimi, Trichoderma reesei, and the like.The genome of the donor microorganism is isolated and cleaved by anappropriate restriction enzyme, such as BamHI. The fragments obtainedare joined to a vector molecule which has previously been cleaved by acompatible restriction enzyme. An example of a suitable vector isplasmid pBR322 which can be cleaved by the restriction endonucleaseBamHI. The amino acid sequence of a cellulase can be used to design aprobe to screen a cDNA or a genomic library prepared from mRNA or DNAfrom cells of interest as donor cells for a cellulase gene.

By using the cellulase cDNA or a fragment thereof as a hybridizationprobe, structurally related genes found in other microorganisms can beeasily cloned. Particularly contemplated is the isolation of genes fromorganisms that express cellulase activity using oligonucleotide probesbased on the nucleotide sequences of cellulase genes obtainable fromCellulomonas fimi. Such probes can be considerably shorter than theentire sequence but should be at least 10, preferably at least 14,nucleotides in length. Longer oligonucleotides are also useful, up tothe full length of the gene, preferably no more than 500, morepreferably no more than 250, nucleotides in length. Both RNA and DNAprobes can be used.

In use, the probes are typically labeled in a detectable manner (forexample with ³² P, ³ H, biotin or avidin) and are incubated withsingle-stranded DNA or RNA from the organism in which a gene is beingsought. Hybridization is detected by means of the label aftersingle-stranded and double-stranded (hybridized) DNA (or DNA/RNA) havebeen separated (typically using nitrocellulose paper). Hybridizationtechniques suitable for use with oligonucleotides are well known tothose skilled in the art.

Although probes are normally used with a detectable label that allowseasy identification, unlabeled oligonucleotides are also useful, both asprecursors of labeled probes and for use in methods that provide fordirect detection of double-stranded DNA (or DNA/RNA). Accordingly, theterm "oligonucleotide probe" refers to both labeled and unlabeled forms.

In order to isolate the cellulose-binding domain of the cellulase,several genetic approaches may be used. One method uses restrictionenzymes to remove a portion of the gene and then to fuse the remaininggene-vector fragment in frame to obtain a mutated gene that encodes aprotein truncated for a particular gene fragment. Another methodinvolves the use of exonucleases such as Bal31 to systematically deletenucleotides either externally from the 5' and the 3' ends of the DNA orinternally from a restricted gap within the gene. These gene deletionmethods result in a mutated gene encoding a shortened protein moleculewhich may then be evaluated for substrate binding ability. Appropriatesubstates for evaluating and binding activity include Avicel, cottonfibres, filter paper, Kraft or ground wood pulp, and the like.

Once a nucleotide sequence encoding the substrate binding region hasbeen identified, either as cDNA or chromosomal DNA, it may then bemanipulated in a variety of ways to fuse it to a DNA sequence encoding apolypeptide of interest. The polysaccharide binding encoding fragmentand the DNA encoding the polypeptide of interest are then ligated. Theresulting ligated DNA may then be manipulated in a variety of ways toprovide for expression. Microbial hosts may be employed which mayinclude, for example bacteria such as E. coli, and eukaryotes such asSaccharomyces cerevisiae.

Preparation of plasmids capable of expressing fusion proteins having theamino acid sequences derived from fragments of more than one polypeptidewith sequence changes when necessary to introduce a convenientrestriction site are described in detail in the experimental section.

Illustrative transcriptional regulatory regions or promoters include,for bacteria, the lac promoter, the TAC promoter, lambda left and rightpromoters, trp and lac promoters, tac promoter, and the like. Thetranscriptional regulatory region may additionally include regulatorysequences which allow the time of expression of the fused gene to bemodulated, for example the presence or absence of nutrients orexpression products in the growth medium, temperature, etc. For example,expression of the fused gene may be regulated by temperature using aregulatory sequence comprising the bacteriophage lambda PL promoter, thebacteriophage lambda OL operator and a temperature-sensitive repressor.Regulation of the promoter is achieved through interaction between therepressor and the operator.

The expression cassette may be included within a replication system forepisomal maintenance in an appropriate cellular host or may be providedwithout a replication system, where it may become integrated into thehost genome. The DNA may be introduced into the host in accordance withknown techniques, such as transformation, using calciumphosphate-precipitated DNA, transfection by contacting the cells with avirus, microinjection of the DNA into cells or the like.

Once the fused gene has been introduced into the appropriate host, thehost may be grown to express the fused gene. In some instances, it maybe desirable to provide for a signal sequence (secretory leader)upstream from and in reading frame with the structural gene, whichprovides for secretion of the fused gene. Illustrative secretory leadersinclude the secretory leaders of penicillinase, immunoglobulins, T-cellreceptors, outer membrane proteins, and the like. By fusion in properreading frame the chimeric polypeptide may be secreted into the medium.

Where the product is retained in the host cell, the cells are harvested,lysed and the product isolated and purified by binding to apolysaccharide substrate. Where the product is secreted, the nutrientmedium may be collected and the product isolated by binding to apolysaccharide matrix. To produce an active protein it may be necessaryto allow the protein to refold.

The recombinant products may be glycosylated or non-glycosylated, havingthe wild-type or other glycosylation. The amount of glycosylation willdepend in part upon the sequence of the particular peptide, as well asthe organism in which it is produced. Thus expression of the product inE. coli cells will result in an unglycosylated product, and expressionof the product in insect cells generally will result in lessglycosylation than expression of the product in mammalian cells.Expression in yeast may result in hyperglycosylation.

In addition to producing fusion proteins from fused genes, the fusionprotein could be made chemically. The substrate binding region ormultiples thereof is produced on its own, purified and then chemicallylinked to the polypeptide of interest using techniques known to thoseskilled in the art.

Use of Fusion Proteins

The subject compositions find a wide variety of applications. Thus thesubject compositions can be used in which recombinant proteins are fusedto the polysaccharide binding region of the cellulase for a generalizedprotein purification technique. The recombinant protein can be readilycleaved from the polysaccharide binding region by the use of a proteasespecific for a sequence present in the cellulose binding region.Examples of biologicals which can be purified in this way includeinterleukin 2, Factor VIII, ligninase, TPA.

The subject compositions can also be used as a means of immobilizing apolypeptide of interest on a cellulosic support, since the substratebinding region adsorption to cellulose is strong and specific. Theimmobilized systems may find a number of uses, including use inpreparing solid state reagents for diagnostic assays, the reagentsincluding enzymes, antibody fragments, peptide hormones, etc.; drugbinding to decrease clearance rate where the cellulose may be eithersoluble, for example carboxymethyl cellulose or a solid support such asa microcrystalline cellulose (Avicel) where the drug is a polypeptidesuch as interleukin 2; drug delivery, for example bound to carboxymethylcellulose and may be used in conjunction with binding of an adjuvant tothe same cellulose support for example for enhancement ofimmunospecificity of the drug to be delivered; dye binding, for examplecoupling of paints or dyes to cellulosic surfaces; printing on forexample paper and cloth (cotton); and to provide hydrolysis or synergy,for example targeting of enzymes such as ligninase for treatment of woodchips, targeting of porphyrins, for example for bleaching of wood pulp;agricultural uses such as binding of insecticides to plant surfaces, forexample BT toxin or other antimicrobials; for nitrogen fixation, forexample for binding of organisms to root surfaces; sustained fertilizerrelease; and sustained release of fungicides; they may also be usedunder conditions of high salt such as in a marine environment foranti-fouling of surfaces exposed to sea water where transfer to freshwater will remove the fusion protein.

Depending upon the particular protocol and the purpose of the reagent,the polypeptide may be labeled or unlabeled. A wide variety of labelshave been used which provide for, directly or indirectly, a detectablesignal. These labels include radionuclids, enzymes, fluoresors,particles, chemiluminesors, enzyme substrates or co-factors, enzymeinhibitors, magnetic particles, etc.

A wide variety of methods exist for linking the labels to thepolypeptides, which may involve use of the end terminal amino group forfunctionalization to form a pyrolezone, while other free amino groupsare protected, where the pyrolezone may then be contacted with variousreagents, for example amino groups, to link to the detectable signalgenerating moiety.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

Abbreviations. pNPC=p-nitrophenyl-β-D-cellobioside; HPA=hide powderazure; gCenA and gCex=the glycosylated forms of CenA and Cex from C.fimi; ngCenA and ngCex=the non-glycosylated forms of CenA and Cex fromrecombinant E. coli; RPC=reverse-phase chromatography; SDS-PAGE=sodiumdodecyl sulfate-polyacrylamide gel electrophoresis; α-Pro/Thr=rabbitantiserum directed against synthetic Cex Pro/Thr box;PMSF=phenylmethylsulfonyl fluoride.

Biological Culture Deposits. A derivative of the cloned gene CenA onplasmid pGEC-2 in Escherichia coli C600 was deposited on Apr. 23, 1986with the 15 American Type Culture Collection (ATCC), 12301 Park LawnDrive, Rockville, Md., 20852, and given ATCC Accession No. 67101. Aderivative of the cloned gene Cex on plasmid pEC-1 was deposited on May29, 1986 and given ATCC Accession No. 67120. E. coli JM83, pUC12-1.1 cexwas deposited on Apr. 23, 1986, and given ATCC excession No. 67102.

EXAMPLE 1 Construction of Cex Expression Plasmids A. Bacterial Strainsand Plasmids

The host strain C600 (thr-1 leu-6 thi-1 supE44 lacyYl tonA21) and theplasmids pcI857 and pCP3 were obtained from Erik Remaut and aredescribed in Gene (1983) 22:103-113.

B. Recombinant DNA Techniques

DNA preparations and enzyme reactions were performed as described byManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, NY. Restriction endonucleases,DNA polymerase I (Klenow fragment), T4 DNA ligase, and the portabletranslation initiation site (PTIS) were purchased from Pharmacia Inc.Bacterial transformations of plasmids containing the leftward promoter(p_(L)) of bacteriophage lambda into strains carrying the cI857 gene ofphage lambda were carried out by the method of Maniatis et al., supra, 1except for the following modification. The bacterial cells were heatshocked at 34° C. for 2 min, incubated in LB medium for 1 hr, and thenplated on selective medium.

C. Growth and Induction of Bacteria

Bacteria were grown in LB (Maniatis, supra) medium with the additionafter autoclaving of 0.4% glucose, 50 μg of kanamycin per ml, and 75 μgof ampicillin per ml. After growth at 30° C. to an optical density at600 nm of 0.3, the cultures were divided, and at 41° C. (induced).

D. Isolation of the cex Gene

The cex gene from C. fimi was isolated as described in U.S. patentapplication Ser. No. 06/859,042, filed May 2, 1986, now abandoned infavor of continuing application Ser. No. 874,292, filed Jun. 6, 1986,now abandoned, and continuing application Ser. No. 630,558, filed Dec.18, 1990, which disclosure is incorporated herein by reference.

E. Plasmid Constructions

1. pUC12-1.1cex.

The cex gene was cloned on a 6.6-kilobase-pair (kbp) BamHI fragment ofC. fimi DNA ligated into the BamHI site of pBR322, giving pEC-1 (FIG.1). The cex gene was localized by deletion analysis to a 2.56-kbpBamHI-SalI DNA fragment yielding pEC-1.1 (FIG. 1). The plasmidpUC12-1.1cex (FIG. 1) contains the 2.56-kbp fragment from pEC-1.1positioned in opposite orientations downstream from thepromoter-operator region of the E. coli lactose operon (lacZp/o) in theplasmid pUC12 (Gene (1982) 19:259-268). The plasmid pEC-1, was describedby Whittle et al., Gene (1982) 17:139-145 and Gilkes et al., J. Gen.Microbiol. (1984) 130:1377-1384, which disclosures are incorporatedherein by reference. The DNA sequence for the RBS, translationalinitiation site, and amino terminus of fusion junctions of βGal-Exgexpression plasmid pUC12-1.1cex are shown below in Table 1.

2. pUC12-1.1(737).

For the construction of pUC12-1.1(737), the 5' untranslated sequences,the ribosome binding site (RBS), and the initiating codon of the cexgene were first removed and replaced with the promoter operator region,the RBS, and the amino terminus of (βGal) from the E. coli lac operonand then with the RBS-ATG sequences of the PTIS. In the first step,pUC12-1.1cex was cut with StyI and BamHI, the staggered ends wererepaired with DNA polymerase I (Klenow fragment), and the plasmid DNAwas ligated under dilute conditions to give pUC12-1.1(737). Thismanipulation results in (i) the in-frame fusion between codon 2 of theCex leader sequence and codon 11 of the alpha-fragment of βGal encodedby pUC12; (ii) the regeneration of the StyI cleavage site; and (iii) thereplacement of the cex initiating codon with a BamHI cleavage site. Thenucleotide sequence and deduced amino acid sequence of the βGal-Cexfusion region of pUC12-1.1(737) are shown in FIG. 2.

3. pUC12-1.1(PTIS).

To obtain pUC12-1.1(PTIS), pUC12-1.1(737) was cut with EcorRI and BamHI,and the 17-bp PTIS with an EcoRI and a BamHI cohesive end was inserted.This procedure resulted in the in-frame fusion of the second codon ofthe cex leader sequence to the initiator ATG of the PTIS. (See FIG. 2)

PTIS

AATTTGGAAAAATTATG

ACCTTTTTAATACCTAG

DNA sequences of the RBS, translational initiation site, and aminoterminus of fusion junctions of βGal-Exg expression plasmids.pUC12-1.1cex codes for unfused cex gene products. The numbering of thecodons of the natural cex gene product in pUC12-1.1cex begins with theinitiating ATG of the leader sequences as -41 and the first codon of themature Exg as +1. The first cex codon in the βGal-Exg fusions retainsits original position number. The deduced amino acid sequence is shownin single-letter code over the DNA sequence. The nucleotides and aminoacids derived from βGal are underlined. Lower-case amino acids are ofnon-lac origin and are derived from the linker region in pUC12. Therestriction sites StyI, AvaII, and EcoRII in the amino terminus of thecex gene were used for fusion of the cex gene to the amino terminus ofβGal in pUC12.

A Exg activity is expressed as nanomoles of p-nitrophenyl released perminute per milligram of total cell protein.

EXAMPLE 2 Construction of CenA Expression Plasmids

A. Bacteria and Medium

E. coli JM101 was used for all cenA experiments. All cultures were grownon LB medium, solidified with 1.5% (w/v) agar when necessary. Ampicillinwas added at a final concentration of 100 μg/ml. CenA activity wasdetected by staining with Congo red after growth of colonies on LBcontaining 1.0% (w/v) agar and 1.0% (w/v) carboxymethyl cellulose (CMC).Liquid cultures were 10 or 50 ml in 50 or 250 ml Erlenmyer flasks; theywere grown in a New Brunswick Gyrotory water bath at 200 rpm.

B. DNA Techniques

Plasmids were released from E. coli by alkaline lysis and purified bycentrifugation to equiliubrium in CsCl-ethidium bromide gradients.Digestion with restriction endonucleases, ligation of fragments andtransformation of E. coli were performed as described.

C. Other Methods

Extracts were prepared by rupturing the cells with a French press.Enzymes were released from the periplasm by osmotic shock. Culturesupernatants were obtained by centrifugation. All enzymes were assayedat 30° C. Endoglucanase from E. coli JM101/pUC18-1.6 cenA was purifiedby immunoadsorbent chromatography, followed by anion exchangechromatography on Mono Q resin with a gradient of 0-1.0 M NaCl in 20 mMpiperazine, pH 9.8. Amino acid sequencing was by automated Edmandegradation using an Applied Biosystems 470A gas-phase sequenator.

D. Isolation of the CenA Gene

The CenA gene from C. fimi was isolated a described in U.S. patentapplication Ser. No. 894,326, filed Aug. 7, 1986, now abandoned in favorof continuing application Ser. No. 630,396, filed Dec. 18, 1990.

E. Plasmid Construction

A 1.6-kb SstI fragment from the 6.0-kb insert of C. fimi DNA in pcEC2was purified and sub-cloned into the SstI site of pUC18 to form pUCEC2,a schematic representation of which is shown in FIGS. 4A and 4B. Theline represents pBR322 DNA; the box is C. fimi DNA; the hatched area isthe cenA coding sequence; the arrow, shows the direction oftranscription; S is SstI; FIG. 4A pcEC2; (FIG. 4B) the nucleotide andamino acid sequences at the fusion point of lacZ and cenA in pUCEC2.

EXAMPLE 3 Construction of Expression Cassette Containing Fusion of cexSBD Gene Fragment and Agrobacterium β-glucosidase Gene abg) andCharacterization of Fusion Protein

A. Construction of Expression Cassette

Plasmid pUC12-1.1cex (PTIS) is cut to completion with PstI. Since boththe vector and the insert have each a PstI restriction site, twopossible fragments are formed. The smaller fragment (approximately 1071bp) is isolated. This DNA fragment corresponds to that portion beginningfrom the PstI site of the insert at nt 1515 to the PstI site of thevector. This PstI-PstI fragment is then completely digested with SphI toproduce three fragments (55 bp, 72 bp and 944 bp). The largest SphI-PstIfragment is isolated.

The larger abg gene fragment (PstI-SphI) and the smaller cex SBDfragment (SphI-PstI) (see FIG. 3) are ligated together in-frame toobtain the desired plasmid construct (approximately 4954 bp). Thisconstruct is called pE01. Plasmid pE01 corresponds to a vector which is2700 bp and the fused cex SBD-abg insert which is 2254 bp. The plasmidconstruct is transformed into E. coli JM101.

B. Enzymactic and PAGE Characterization of the Fusion Protein

The fusion protein encoded by pE01 is characterized for its catalyticactivity compared to the original Abg and for its ability to bind toAvicel compared to the original Cex. Characterization of catalyticactivity includes determination of the kinetic properties (e.g., K_(m)and V_(max)) and substrate specificity of the fusion enzyme. Enzymeactivity is determined by the amount of glucose produced from a fixedconcentration of cellobiose under standard assay time, temperature, pH,ionic strength and buffer. The glucose concentration is measured using aglucose analyzer (Beckman). The analysis is based on the initial rate ofoxygen consumption in the conversion glucose to gluconic acid asdetermined by an oxygen electrode; the rate of oxygen consumption isdirectly proportional to the amount of glucose present relative to aknown standard glucose solution.

The fusion protein is also analyzed by SDS-PAGE to determine relativemolecular mass. The purified fusion protein can be cleaved with theprotease from C. fimi to produce two or more other protein fragments.This is ascertained by running an SDS-PAGE of a proteolytic cleavagemixture of the fusion protein and doing a zymogram using a fluorescentglucoside derivative, MUG (4-methylumbelliferyl-β-D-glucoside) or X-glu(5-bromo-4-chloro-3-indolyl-λ-D-glucopyranoside). This will alsodetermine whether other smaller active enzyme fragments are formed andtheir relative sizes.

C. Characterization of the Adsorption Properties of the Fusion Enzyme

Adsorption of cellulase to cellulose is presumed to be the first steprequired in the hydrolysis of insoluble cellulosic substrates. Enzymebinding to cellulose has been investigated for a few microbialcellulases with the aim of understanding how factors like enzymeconcentration, enzyme, combination and ratio, temperature, pH and ionicstrength of buffer might affect the adsorption kinetics of cellulase andthe rate of cellulose degradation (Ghose & Bisaria, 1979; Moloney &Coughlan, 1983; Ooshima et al., 1983; Ryu et al., 1984; Andrease et al.,1987; Willaimson & Stutzenberge, 1987).

The ability of the fusion enzyme to bind to cellulosic substrate isanalyzed by calculation of the adsorption equilibrium constant (K_(a)).Previous studies have shown that the adsorption of cellulase tocellulose follows the Langmuir isotherm equation (Langmuir, 1916):##EQU1## where C_(b) is the amount of enzyme bound per unit weight ofcellulose at equilibrium, C_(f) is the free enzyme concentration,C_(max) is the maximum adsorption amount of enzyme and K_(a) is theadsorption equilibrium constant. From equation (1), a more usefulequation (2) is derived which can be plotted easily to obtain the valuesof K_(a) and C_(max). This is given as: ##EQU2## Equation (2) is used toplot C_(f) /C_(b) against C_(f) to obtain a straight line according tothe equation y=mx+b. The slope (m) is given by 1/C_(max) and theintercept (b) is given by 1/K_(a) C_(max). The values obtained for K_(a)and C_(max) are important in that they measure the adsorption affinityof the enzyme to the substrate and the number of adsorption sites perunit surface of the adsorbent, respectively. The K_(a) value inparticular is needed so that meaningful comparisons of the effects ofdifferent physical and chemical parameters on the adsorption of thefusion enzyme to cellulose can be made.

The ability of the enzyme to bind to Avicel is expressed as thepercentage enzyme bound relative to the known activity concentration ofthe enzyme introduced into the system, of the free enzyme present in thesupernatant fluid and of the bound enzyme eluted from substrate withdistilled water. Kinetic studies on the adsorption process of the enzymetowards cellulosic substrate at varying enzyme concentration includesthe determination of K_(a) at different pH, temperature and ionicstrength of the buffer. Stability (operational and storage) of theimmobilized fusion protein is determined by binding the enzyme to Avicelin batch or column and allowing enzymatic reaction to occur as afunction of time. The amount of glucose recovered, the activityconcentration of the fusion protein and the amount of protein in theeluent versus time will indicate the stability of the immobilizationscheme.

EXAMPLE 4 Isolation of DNA Fragment Responsible for Substrate Binding

To define the specific SBD peptide involved in substrate binding,several genetic approaches are available. One method uses restrictionenzymes to remove a portion of the gene and then to fuse the remaininggene-vector fragment in-frame to obtain a mutated gene that encodes aprotein truncated for a particular gene fragment. Another methodinvolves the use of exonucleases (e.g., Bal31) to systematically 1delete nucleotides either externally from the 5' and the 3' ends of theDNA or internally from a restricted gap within the gene. These genedeletion methods have the ultimate goal of producing a mutated geneencoding a shortened protein molecule, whose function may or may not bethe same as the original protein molecule. Alteration of function in thetruncated protein may be as a result of either the removal of thatparticular peptide fragment per se or from conformational changes in themodified protein as a result of deletion of some amino acids.

A. Deletion Using XmaIII Restriction Enzyme

The plasmid pUC12-1.1cex (PTIS) is shown by FIG. 6 with the relevantrestriction sites and sizes.

Initial binding studies of a SalI (S) partial digest of the plasmideliminating that portion of the gene between nucleotide (nt) 1962 and nt2580 have shown that the resulting truncated protein did not bind toAvicel. This result does not prove that the peptide encoded between theSalI site (nt 1962) to the stop codon (TGA at nt 2189) is the essentialregion for binding of the enzyme. The region just before the start ofthe deletion could have well been an important region for binding tocellulose. Another factor that could have contributed to the nonbindingto cellulose by the SalI deletion mutant is the formation of a fusionprotein between the deleted Cex and the β-galactosidase of the vector.

Assuming an amino acid has an average molecular weight of 110, thedeleted peptide in the SalI mutant is approximately 8 kD in size. Thispredicted size corresponds well to the size of a peptide that waspurified by FPLC (Pharmacia) from a sample of proteolytically cleavedexoglucanase and that was subsequently found to bind tightly to Avicel.This result strongly suggests that the specific SBD peptide is withinthis apparent 8 kD region. The N-terminus of the FPLC purifiedapproximate 8 kD peptide has been sequenced to determine exactly wherethe proteinase cleavage site is. Results indicate that the amino acidcleavage site occurs at the end of the PT box (between the lastthreonine and serine). Based on this amino acid sequence result, thecalculated size of the SBD peptide should have been 11.3 kD. Thisdiscrepancy between the size of the FPLC purified SBD peptide and thecalculated size as predicted from the amino acid cleavage site couldhave arisen from an aberrant migration of the peptide on thepolyacrylamide gel.

To delineate further the amino acid sequence involved in substratebinding, the plasmid pUC12-1.1cex is digested partially with XmaIII (seeTable 3). The linearized fragment corresponding to 5107 bp in size isisolated, religated and transformed into E. coli JM101. The gene portionbetween nt 1873 and nt 2074 is deleted and the remaining gene-vector isfused back together in-frame. The truncated protein produced and itsbinding affinity for Avicel is characterized and compared to theoriginal cex protein.

B. Deletion Using Bal31

Bal31 is a highly specific nuclease that simultaneously degrades boththe 3' and 5' ends of dsDNA without internal single-stranded cuts. Sincethere is an absolute requirement of the enzyme for Ca⁺⁺, the extent ofdeletion by the enzyme can be monitored and controlled by simply addinga divalent chelating agent, EGTA to the reaction mixture (Maniatis etal., 1982).

Before submitting the cex gene to Bal31 digestion, a loopout fragmentcontaining the following regions is synthesized: 1) a restriction sitewhere deletion will start (XbaI which is only found in the vector andjust a few nucleotides downstream of the C. fimi gene insert); 2) asecond restriction site not found in either the vector or the insert(NcoI); 3) a stretch of nucleotides containing stop codons in all threereading frames.

The loopout fragment is first annealed to a M13 ssDNA templatecontaining the insert. The fragment is extended by adding d(A,T,G andC)TPs, Klenow polymerase and ligase. This fragment is transformed into Ecoli JM101 and the plaques hybridizing with the labeled loopout primerare picked up. The replicative form of DNA is isolated from the E. colitransformants. The duplex DNA is first cut with XbaI to linearize DNA.The same linearized DNA is then cut with NcoI. A stuffer DNA fragmentcontaining C. fimi DNA flanked at one end with an NcoI site is also cutwith NcoI. The stuffer DNA is ligated to the linearized DNA toregenerate an NcoI site. This construct is then digested with Bal31which will digest from both ends (in the stuffer DNA and in the cex geneinsert) at almost the same rate. The reaction mix is stoppedperiodically by removing a portion of the reaction sample and putting itinto DNA buffer containing EGTA to stop Bal31 digestion. The stuffer DNAis removed by adding NcoI to the inactivated Bal31-digested DNA mixture.The DNA is then filled in with Klenow polymerase, size fractionated inan agarose gel and bluntligated to pUC12 to obtain a closed, circular,duplex DNA. A few microliters from the ligated mix is then cut with tworestriction enzymes in such a way that small differences in the insertlength as a result of deletion by Bal31 can easily be ascertained. TheDNA is transformed into competent E. coli JM101 cells. To screen for afamily of mutants deleted at the 3' end of cex, antibody raised againstthe apparent 8 kD SBD peptide is used to identify positive deletionclones.

Truncated proteins produced from the different deletion mutants aretested for their ability to bind to Avicel and for their catalyticactivity as described above.

EXAMPLE 5 Production of Glucose from Cellobiose Using β-glucosidaseFusion Protein Immobilized on Avicel

This procedure uses endoglucanase-exoglucanase co-incubation withsubsequent channeling of the resulting cellobiose mixture into an Avicelcolumn immobilized with β-glucosidase. The method is as follows. In afermentation vessel, a suitable proportion of both endoglucanase andexoglucanase is added to a medium containing the cellulosic material tobe degraded. The enzymes are allowed to react for a fixed period of timeto produce cellobiose, which is solubilized in the medium. The wholespent medium together with the enzyme is first passed through an Avicelcolumn which will immobilize and concentrate both the endoglucanase andthe exoglucanase. The eluent containing the cellobiose is channeled to asecond column immobilized with β-glucosidase fusion protein which thenhydrolyzes the cellobiose into glucose units. The endoglucanase and theexoglucanase are regenerated from the first column by simply elutingthem out with distilled water. Both columns can be reused several timesfor purification and enzymatic conversion.

EXAMPLE 6 Preparation of cenA-alkaline Phosphatase Fusion ProteinExpression Cassette

TnphoA is a derivative of transposon Tn5 containing the E. coli alkalinephosphatase gene, phoA, minus its signal sequence ('phoA).Transpositional insertion into an expressed gene in the correct readingframe creates a PhoA fusion protein. If the target gene contains proteinexport signals, these can direct the secretion of the fusion protein.This secretion is detectable by alkaline phosphatase activity, which ispresent only when the enzyme has a periplasmic location. TnphoA is usedto create phoA gene fusions with the C. fimi cenA gene in a plasmidhaving a multiple cloning site. A gene encoding a protein of interestcan be cloned into the multiple cloning site and expressed as a fusionprotein. The gene product is purified by binding to cellulose, such asAvicel, and cleavage from the CenA fusion partner with C. fimi protease

A. Preparation and Analysis of Gene Fusions

Transpositional mutagenesis with TnphoA is used to create gene fusionswith cenA. The plasmid containing cenA is pUCEC2, a 1.6 kb SstI cenAfragment cloned in pTZ18U, a multifunctional derivative of pUC18(Yanisch-Perron et al., Gene (1985) 33:103-119). pTZ18U is availablefrom U.S. Biochemicals.

Oligonucleotide-directed matagenesis (Zoller et al., Nucleic Acids Res.(1982) 10:6487-6500 and Zoller et al., Methods Enzymol. (1983)100:468-500) was used to delete the carboxy-terminal portion of the cenAgene and juxtapose the Pro-Thr box and the multiple cloning site ofpTZ18U. Screening procedures include dot blot hybridization using themutagenic oligonucleotide as a probe, and restriction analysis. DNAsequencing by the chain-termination method was performed to verify thesequence of the deletion region (Yanisch-Perron, supra).

The transposition event was mediated by infection of E. coli CC118(pUCEC2) with a defective lambda phage containing the transposon,λTnphoA-1 (Gutierrez et al., J. Mol. Biol. (1987) 195:289-297). E. coliCC118 contains a deletion in the phoA gene. Transpositional insertioninto the cenA gene in-frame with CenA the periplasm, secretion beingpromoted by the CenA signal peptide. Colonies selected for kanamycin(transposon-derived) and ampicillin resistance were screened foralkaline phosphatase activity on the indigogenic substrate5-bromo-4-chloro-3-indolyl phosphate (XP). Plasmid DNA from PhoA+colonies was retransformed, and selected and screened as above.PhoA+colonies were screened for endoglucanase activity oncarboxymethylcellulose (CMC) plates stained with Congo red (Gilkes etal., Bio/Technology (1984) 2:259-263). The desired penotype is PhoA+,Eng-, and resistance to ampicillin and kanamycin.

Plasmid DNA was isolated from PhoA+, EngA-colonies and analyzed byrestriction digestion and agarose gel electrophoresis. Of 55 coloniesscreened, 34 had TnphoA insertions in cenA in the correct orientation.The insertions occurred throughout the cenA gene. Some of these clonesmay have out-of-frame insertions, a possibility that will become evidentwhen looking at the protein products of the fusions. Analysis ofcellulose binding of some of the CenA-PhoA fusion proteins shows thatthe fusion proteins bind to filter paper, despite stringent washes with50 mM phosphate buffer (pH 7.0) and 0.5 M NaCl.

One fusion protein which binds to cellulose is selected for furtherstudy. The exact insertion position of TnphoA is determined by DNAsequencing using the chain-termination method. The buffer conditionswhich facilitate binding to Avicel and for which elution from Aviceloccurs are also determined as described above (see Example 3).

The Avicel-bound fusion protein is incubated with C. fimi protease, andreleased proteolytic fragments are concentrated by ultrafiltration andanalyzed by SDS-PAGE and PhoA activity zymogram or Western immunoblot,or by gel filtration chromatography. Substrate-bound fragments aredissolved in SDS and analyzed by SDS-PAGE and Western immunoblot, probedwith antiserum to the Pro-Thr box (Langsford et al., FEBS Letters (1987)225:163-167).

B. Purification of Fusion Protein

Cleared E. coli cell extracts containing the fusion protein are appliedto an Avicel column in a buffer which promotes binding of the fusionprotein to the Avicel matrix. After thorough washing of the column withbuffer to remove non-specifically bound proteins, C. fimi protease isapplied to the column and washed through with buffer. Collectedfractions are assayed for alkaline phosphatase activity, and the enzymepeak further purified by ion exchange or gel filtration chromatography.Purification conditions, such as protease concentration and flowrate,are varied to optimize the recovery of alkaline phosphatase activity.

EXAMPLE 7 Use of Cellulomonas fimi Cellulose Binding Domains for DrugDelivery

A. Solubility/Persistence Interleukin 2

A fusion protein comprising interleukin 2 (IL-2) linked to the cellulosebinding region of a C. fimi cellulase is prepared as described above bypreparing a fusion gene comprising at least the DNA sequence encodingthe CenA or Cex cellulose binding region and a gene encoding IL-2 or afunctional portion thereof and transforming it into an expression hostsuch as E. coli. The fusion protein is purified by affinitychromatography on cellulose (Avicel or cotton). The fusion protein iseluted with water and then bound to soluble (carboxymethyl) or insoluble(Avicel) cellulose. These conjugates are injected into mice (i.p.) andthe kinetics of IL-2 clearance from the peritoneal fluid determined. Thesoluble conjugate is injected i.v. and the kinetics of clearance of IL-2activity from the blood determined. The conjugates find use indecreasing the clearance rate of IL-2 from the circulation.

B. Antigenicity/Adjuvant Activity

Two fusion proteins comprising IL-2 and alkaline phosphataserespectively linked to the cellulose binding region of C. fimicellulase, prepared as described above, are bound to the same cellulosepreparation through the cellulose binding region on each fusion protein.Both soluble (for example, carboxymethyl) and insoluble (for example,Avicel) cellulose matrices are used. The combined matrixIL-2-CBR→cellulose←CBR-alkaline phosphatase is injected into mice andthe immune responses (T-cell proliferation and anti-alkaline phosphataseantibody concentration) determined after 1 week and 2 weeks. Theseresponses are compared to the response generated by injecting anidentical amount of alkaline phosphatase-CBR. In subsequent experimentsHIV gp 120-CBR and Pseudomonas porin-CBR are tested in an analogoussystem replacing alkaline phosphatase. The combination of IL-2 in closeproximity to an antigen finds use in enhancing the immune response tothe presented antigen.

The compositions of the subject invention comprise hybrid proteins inwhich at least the polysaccharide binding domain of a polysaccharidaseis fused to a polypeptide of interest. The compositions find use forbinding a variety of ligands to a polysaccharide matrix, either solubleor insoluble. They may be used bound to the matrix, for example as drugdelivery systems, or in fermentors, or they may be used as a means ofisolating or purifying the ligand, then recovering the ligand followingcleavage with a specific protease.

All publications and patent applications mentioned in this specificationare indicative of the i level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. In a method using an affinity matrix forimmobilization of a polypeptide of interest, the improvement whichcomprises:preparing a fusion protein comprising said polypeptide ofinterest and an amino acid sequence comprising a substrate bindingregion of a cellulose with the provisio that said amino acid sequence isessentially lacking in cellulose activity; and contacting said fusionprotein with an affinity matrix comprising a cellulose substrate forsaid cellulose whereby said substrate binding region binds to saidaffinity matrix.
 2. A method according to claim 1, wherein saidpolypeptide of interest is a β-glucosidase or interleukin-2.
 3. In amethod using an affinity matrix for purification of a polypeptide ofinterest, the improvement which comprises:preparing a fusion proteincomprising said polypeptide of interest and an amino acid sequencecomprising a substrate binding region of a cellulose, with the provisothat said amino acid sequence is essentially lacing in celluloseactivity; contacting said fusion protein with an affinity matrixcomprising a cellulose substrate for said cellulose whereby saidsubstrate binding region binds to said affinity matrix; and separatingthe fusion protein or the polypeptide of interest from the affinitymatrix.
 4. The method according to claim 3, wherein said substratebinding region is contained by cellulase is obtained from Cellulomonasfimi.
 5. The method according to claim 4, wherein said substrate bindingregion is contained by cellulase obtained from Cellulomonas fimi.
 6. Themethod according to claim 3, wherein said affinity matrix ismicrocrystalline cellulose.
 7. The method according to claim 3, whereinsaid polypeptide of interest is an alkaline phosphatase.